EP3145540A2 - Chemically activated nanocapsid functionalized for cancer targeting - Google Patents
Chemically activated nanocapsid functionalized for cancer targetingInfo
- Publication number
- EP3145540A2 EP3145540A2 EP15796952.8A EP15796952A EP3145540A2 EP 3145540 A2 EP3145540 A2 EP 3145540A2 EP 15796952 A EP15796952 A EP 15796952A EP 3145540 A2 EP3145540 A2 EP 3145540A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- hev
- capsid protein
- vlp
- modified capsid
- cysteine
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5107—Excipients; Inactive ingredients
- A61K9/513—Organic macromolecular compounds; Dendrimers
- A61K9/5169—Proteins, e.g. albumin, gelatin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/525—Virus
- A61K2039/5258—Virus-like particles
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/28011—Hepeviridae
- C12N2770/28111—Hepevirus, e.g. hepatitis E virus
- C12N2770/28122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/28011—Hepeviridae
- C12N2770/28111—Hepevirus, e.g. hepatitis E virus
- C12N2770/28123—Virus like particles [VLP]
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/28011—Hepeviridae
- C12N2770/28111—Hepevirus, e.g. hepatitis E virus
- C12N2770/28141—Use of virus, viral particle or viral elements as a vector
- C12N2770/28142—Use of virus, viral particle or viral elements as a vector virus or viral particle as vehicle, e.g. encapsulating small organic molecule
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2810/00—Vectors comprising a targeting moiety
- C12N2810/40—Vectors comprising a peptide as targeting moiety, e.g. a synthetic peptide, from undefined source
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
Definitions
- Vims-like particles can serve as nanocarriers for targeted delivery of diagnostics and therapeutics regimes, such as DNA/RNA and a variety of chemotherapeu ics.
- Hepatitis E virus HEV
- HEV vims-like particles are capsid protein ieosahedral cages that can be produced by expression of the major capsid protein HEV Open Reading Frame 2 (ORF2) in a eukaryotic expression system.
- HEV VLPs are stable in acid and proteolytic
- HEV VLPs represent a promising nano-carrier that can be exploited, e.g., for
- the present invention provides a cysteine modified HEV V LP scaffold for altering HEV VLP surface functionality.
- the present invention provides a modified capsid protein comprising a portion of hepatitis E virus (HEV) open Reading Frame 2 (ORF2) protein that is able to form an acid and proteolyticaliy stable HEV virus like particle (VLP), wherein: the portion of HEV ORF2 comprises a P-domain of the HEV ORF2 protein; the P-domain comprises at least one surface variable loop and a C -terminus; the P-domain comprises a cysteine in the at least one surface variable loop or at the C-termmus; and the HEV ORF2 portion retains its ability to form an acid and proteolyticaliy stable HEV VLP when the surface variable loop or C-terminal cysteine is chemically derivatized.
- HEV hepatitis E virus
- ORF2 open Reading Frame 2
- VLP acid and proteolyticaliy stable HEV virus like particle
- the HEV ORF2 portion retains its ability to form an acid and proteolyticaliy stable HEV VLP when the surface variable loop or C-terminal cysteine is alkylated, acylated, arylated, succinylated, oxidized, or conjugated to a detectable label or bioactive agent.
- the modified capsid protein comprises an amino acid sequence at least 90%, 95%, or 99% identical, or identical, to residues 112-608 of the HEV ORE 2 protein of SEQ ID NO: 1, 2, 3, 4, 5, or 6.
- the at least one P- domain surface variable loop or C-terminal cysteine is conjugated to a detectable label.
- the detectable label comprises a fluorophore, a superparamagnetic label, an MRI contrast agent, a positron emitting isotope, or a cluster of elements of group 3 through 18 having an atomic number greater than 20.
- the cluster of elements of group 3 through 18 having an atomic number greater than 20 comprises a gold nanocluster.
- the at least one P-domain surface variable loop or C-terminai cysteine is conjugated to a bioactive agent.
- the bioactive agent is a heterologous peptide.
- the heterologous peptide is a ceil targeting ligand.
- the cell targeting ligand is a cancer cell targeting liganc!. In some embodiments, the cancer ceil targeting ligand is LXY30. In some embodiments, the cancer cell targeting ligand is an antibody that binds an antigen expressed on the surface of a cancer cell. In some embodiments, the modified capsid protein further comprises a second cysteine in a P-domain surface variable loop or at the C-termimis of the P-domam. In some embodiments, the second cysteine is conjugated to a chemotherapeutic.
- the second cysteine is conjugated to a detectable label.
- the detectable label conjugated to the second cysteine comprises a fluorophore, a superparamagnetic label, an MRI contrast agent, a positron emitting isotope, or a cluster of elements of group 3 through 18 having an atomi c number greater than 20.
- the detectable label conjugated to the second cysteine comprising the cluster of elements of group 3 through 18 having an atomic number greater than 20 comprises a gold nanoc luster.
- the at least one P-domain surface variable loop cysteine is alkylated, acylated, arylated, succinylated, or oxidized.
- the at least one P-domain surface variable loop cysteine of HEV ORF2 replaces Y 85, T489, S533, N573, or T586 of HEV ORF2 or the C-terminal cysteine replaces residue 608 of HEV O F2.
- the modified capsid protein is a component of an acid and
- the acid and proteolytically stable HEV V LP encapsulates a bioactive agent.
- the encapsulated bioactive agent is a heterologous nucleic acid, a heterologous peptide, a detectable label, a non-protemogenic amino acid, an oligosaccharide, a synthetic macromolecule, or a chemotherapeutic.
- the encapsulated detectable label comprises a fluorophore, a superparamagnetic label, an MRI contrast agent, a positron emitting isotope, or a cluster of elements of group 3 through 18 having an atomic number greater than 20.
- the present invention provides a composition comprising the modified capsid protein of any one of the foregoing aspects or embodiments and a pharmaceutically acceptable excipient.
- the composition comprises an HEV VLP having at least one cysteine within an HEV ORF2 P-domain surface variable loop or C-terminus that is chemically conjugated to a cell targeting ligand, a bioactive agent, or a detectable label. The chemical conjugation can result in a heterologous arrangement between the cysteine and the cell targeting ligand, bioactive agent, or detectable label.
- the present invention provides a nucleic acid (e.g., isolated nucleic acid) comprising a polynucleotide sequence encoding any one of the foregoing modified capsid proteins.
- the present invention provides an expression cassette comprising a promoter (e.g., heterologous promoter) operably linked to a polynucleotide sequence encoding an one of the foregoing modi fied capsid proteins.
- the present invention provides a cell (e.g. , isolated cell or host cell) comprising the foregoing nucleic acid or the foregoing expression cassette.
- the present invention provides a cell (e.g., isolated cell or host cell) comprising any one of the foregoing modified capsid proteins.
- a cell e.g., isolated cell or host cell
- the present invention provides an organism comprising any one of the foregoing modified capsid proteins.
- the present invention provides a method of producing a modified capsid protein comprising cultivating any one of the foregoing cells, isolated cells, or host cells under conditions suitable to permit expression of the modified capsid protein.
- the method further comprises purifying the capsid protein.
- the method further comprises derivatizing the at least one P-domain surface variable loop or C-terminal cysteine.
- the derivatizing comprises acylating, alkylating, arylating, succinylating, or oxidizing the P-domain surface variable loop or C-terminal cysteine.
- the derivatizing comprises conjugating a bioactive agent to the at least one P-domain surface variable loop or C-terminal cysteine.
- the present invention provides a method of directing an HE V VLP to a target cell comprising contacting a cell with the HEV-VLP, wherein the HEV VLP comprises any one of the foregoing modified capsid proteins, wherein the HEV VLP further comprises a cell targeting moiety having affinity for the target cell conjugated to the at least one P-domain surface variable loop or C-terminal cysteine, in some embodiments, the HE V VLP further comprises a detectable label, and the method further comprises detecting the detectable label.
- the detecting the detectable label comprises detecting a fluorophore, a superparamagnetic label, an MRI contrast agent, a positron emitting isotope, or a cluster of elements of group 3 through 18 having an atomic number greater than 20.
- the detecting the cluster of elements of group 3 through 18 having an atomic number greater than 20 comprises detecting a gold nanocluster.
- the cell targeting moiety having affinity for the target cell conjugated to the at least one P-domain surface variable loop or C-terminal cysteine is a heterologous peptide comprising a cancer cell targeting ligarsd, and the target cell is a cancer cell.
- the HEV VLP or a component thereof enters the cancer cell.
- the HEV V I J' encapsulates a hioactive agent, and the method comprises delivering the bioactive agent to the intracellular region of the cell.
- the encapsulated bioactive agent is a heterologous nucleic acid, a heterologous peptide, a detectable label, a non-proteinogenic amino acid, an oligosaccharide, a synthetic
- the encapsulated detectable label comprises a fluorophore, a superparamagnetic label, an MRI contrast agent, a positron emitting isotope, or a cluster of elements of group 3 through 18 having an atomic number greater than 20.
- the encapsulated cluster of elements of group 3 through 18 having an atomic number greater than 20 comprises an encapsulated gold nanocluster.
- Figure 1 (A) Depicts the location of surface accessible flexible coil regions (P- domain surface variable loops) of HEV ORF 2, and residues Y485, T489, S533, N573, and T586, which lie within such flexible coil regions and were selected for cysteine mutation. (B) All the selected residues for cysteine mutation were located at the surface of the P- domain, with residue Y485, T489, T586 on the outmost surface, and residues S533 and N573 at side facing the VLP 5-fold axis.
- FIG. 2 Characterization of chimeric HEV VLP carrying a cysteine replacement mutation.
- A The cysteine mutation at 573 appeared to allow efficient biotinylation of the chimeric VLP as compared to the other mutation sites, i.e., Y485, T489, S533, and T586, although those mutations exhibited similar biotinylation after VLP disassembly
- B Both the wild type VLP and the chimeric VLP had similar binding affinity to monoclonal antibody HEP40-4B, while chimeric VLP exhibited decreased reactivity to monoclonal antibody HEP230.
- Figure 3 (A) The three-dimensional density map of HEV wt-VLP in complex with Fab fragments of HEP230 is displayed as isosurface along a 5-fold axis, showing extra density (arrow) in the gap of two 5-fold related protruding dimers. (B) The density agreed with the footprin t of the Fab molecule. (C) Residue N573 (sphere mode) is located next to the binding site of HEP230 that (D) allows only one Fab molecule binding per 5-fold region.
- FIG. 4 The schematic of the two-steps chemical conjugation process including a thiol selective reaction and a Copper Catalyzed Azide-Alkyne Cycloaddition (CuAAC) or "click chemistry" reaction to form LXY30-VLPs.
- CuAAC Copper Catalyzed Azide-Alkyne Cycloaddition
- Method 1 (A) The Copper Catalyzed Azide-Alkyne Cycloaddition (CuAAC) reaction between Mal-Azide and Alkyne-LXY3() to form maleimide linked LXY30 (Mat- LXY30) was done first.
- Method 2 (A) The conjugation process starting by labeling maleimide linked azide (Mal-Azide) at Cys sites of N573C-VLPs through Thiol selective-reaction to build azide linked VLPs (Azide-VLPs), followed by adding LXY30- Alkyne, ascorbic acid and CuS04 to form LXY30 linked VLPs (LXY30-VLPs).
- FIG. 1 Damaged or dis-assembled LXY30-VLPs resulting from the conjugation process, due to the presence of reactive agents including azide, ascorbic acid and CuS04, can be separated from intact VLPs by, e.g., centrifugation, size exclusion chromatography, and the like.
- Figure 5 In vitro fluorescence data of VLP-Cy5.5 and LXY30-VLP-Cy5.5 binding to MDA-MB-231 breast cancer ceils.
- FIG. 6 The LXY30 chemical structure and its binding to U-87MG cells. Both scrambled LXY30 and LXY30 were directly conjugated to FITC, incubated with U-87MG cells, and detected by flow cytometry. Compared with the negative control, LXY30 showed obvious binding to U-87MG cells, which highly express alpha3-betal-integrin. The scrambled LXY30 showed minimum uptake due to non-specific staining from the fluorophore FiTC.
- Figure 7 In vivo NIR fluorescence images of real-time tumor targeting
- Forward 5 '-3' and Reverse 3 '-5 ' primers were designed with a cysteine coding sequence to the replace parental amino acid, a-e represent Y485C, T489C, S533C, N573C, and T586C respectively.
- J Figure 9 Depicts the location of the surface variable loops and C -terminal residues of the P-domain of the HEV ORF2 capsid protein.
- A Depicts a schematic diagram of P- domain surface variable loops of HEV ORF 2 capsid protein.
- B Depicts the location of the P-domain surface variable loop residues and C-terminal residue on an HEV VLP.
- One or more cysteines can be positioned within the indicated loop or C-terminal residues for conjugation to a molecular payload.
- FIG. 10 Schematic depicting the application of chimeric HEV ORF2 VLPs as nanocarriers for drug delivery.
- the periodicity of the capsid, the presence of surface- accessible amino acids with reactive moieties, and the tolerance of a single chain version of the capsid protein dimer to diverse peptide insertions enable dense, repetitive display of targeting iigands either by chemical conjugation or genetic insertion, and display of aptamers, glycoproteins, etc., by chemical conjugation.
- HEV ORF2 VLPs also possess relatively large interior volumes that can be loaded with a variety of materials, including DNA plasmids. In comparison to other virus-like particles, HEV ORF2 VLPs possess strong structural plasticity, with a stability switch sensitive to calcium concentration.
- FIG. 11 Cys-VLP conjugation to sulfur protected Auio 2 .
- A Depicts two methods for covalent gold conjugation to Cys-VLP.
- B Cys-VLP direct ligand exchange with Auio 2 (pMBA) 4 4.
- C Cys-VLP maieimide conjugation with Au ⁇ -Ce-Maleimide.
- D Cryo-EM Cys-VLP with A io2(pMBA) 4 a d the processed 3-D reconstruction,
- Figure ⁇ 2 Schematic depicting the process used to encapsulate quantum dots in an HEV ORF2 VLP (top). Transmission electron microscope image of ferrite packaged VLPs showing darker intensity compared to package-free VLPs. DETAILED DESCRIPTION OF THE INVENTION I. Definitions
- Hepatitis E virus refers to a vims, virus type, or virus class, which i) causes water-borne, infectious hepatitis; it) is distinguished from hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C vims (HCV), or hepatitis D vims (HDV) in terms of serological characteristics: and iii) contains a genomic region that is homologous to a 1.33 kb cD ' NA inserted in pTZ Fl (ET1.1), a plasmid embodied in a E. coli strain deposited in American Type Culture Collection (ATCC) with accession number 67717.
- HAV hepatitis A virus
- HBV hepatitis B virus
- HCV hepatitis C vims
- HDV hepatitis D vims
- capsid protein and “modified capsid protein,” with reference to HEV, refer to a mature or modified (e.g. , truncated, recombinantly mutated, or chemically derivatized) HEV open reading from 2 (ORF2) polypeptide.
- ORF2 polypeptides or proteins refer to include the full-length polypeptide, and fragments thereof, and also include any substitutions, deletions, or insertions or other modifications made to the ORF2 proteins.
- the capsid proteins must he capable of forming a virus like particle (VLP).
- VLP virus like particle
- the capsid protein contains at least residues 112-608 of HEV ORF2, although the capsid protein can tolerate various additional substitutions, deletions, or insertions so long as they are tolerated without abrogating VLP formation.
- the term "modified capsid protein” refers to a capsid protein, or portion thereof, in which a surface variable loop of the P-domain of HEV ORF2 is modified to incorporate one or more cysteines that are not otherwise present in the wild-type capsid protein sequence.
- modified capsid protein refers to a capsid protein, or portion thereof, in which the C-terminus (e.g., position 608) of HEV ORF2 is modified to incorporate one or more cysteines that are not otherwise present in the wild-type capsid protein sequence.
- modified capsid protein refers to a capsid protein, or portion thereof, in which a cysteine (e.g. , a cysteine of a surface variable loop of the P-domain of H EV ORE 2 or a cysteine recombinantly introduced at position 608) is chemically derivatized to covalentlv conjugate to the protein at least one heterologous atom or molecule.
- the cysteine can be inserted such that the HEV ORF2 protein length is increased, or the cysteine can replace one or more residues of a P-domain surface variable loop and/or C-terminus.
- modified capsid proteins retain the ability to form HEV VLPs.
- the one or more cysteines are conjugated to a bioactive agent (e.g., a cell-targeting ligand such as the peptide LXY30).
- P-domain surface variable loops include one or more of , e.g., residues 475-493; residues 502-535; residues 539-569; residues 572-579; and residues 581-595 of HEV ORF 2 (SEQ ID NO: l, 2, 3, 4, 5, or 6).
- P-domain surface variable loops further include the residues of polypeptides comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 99%, or more identical to one or more of SEQ ID NOS:l, 2, 3, 4, 5, or 6 and that correspond to one or more of residues 475-493; residues 502-535; residues 539-569; residues 572-579; and residues 581 -595 of SEQ ID NOS: l, 2, 3, 4, 5, or 6.
- VLP virus-l ike particle
- VLP refers to an icosahedrai shell (e.g., Tl or T 3) formed by a capsid protein.
- VLPs are not infectious due to the lack of a viral genome.
- VLP refers to a nonreplicating icosahedrai viral shell, derived from hepatitis E vims capsid protein HE V ORF2, a portion thereof. VLPs can form spontaneously upon recombinant expression of the protein in an appropriate expression system.
- the VLP is formed from a modified capsid protein, e.g., a capsid protein containing one or more cysteine residues in a surface variable loop of HEV ORF2, or a portion thereof.
- An HEV VLP can contain a mixture of modified and/or unmodified HEV ORF2 proteins.
- an acid e.g., pH of, or of about, 6, 5.5, 5, 4.5, 4,
- a population of HEV VLPs (e.g., modified or unmodified) can be incubated under acid and/or proteolytic conditions for a suitable period of time (e.g., for at least, or for at least about, 1 , 2, 3, 4, 5, 10, 15, 20, 30, 45, or 60 minutes) and then tested to determine the extent of quaternary structure retention.
- a suitable period of time e.g., for at least, or for at least about, 1 , 2, 3, 4, 5, 10, 15, 20, 30, 45, or 60 minutes
- HEV VLP refers to a modified HEV VLP that when incubated as a population of VLPs under acid and/or proteolytic conditions and assayed by electron microscopy, at least 10%, 25%, 50%, 75%, 90%, 95%, 99%, or 100% of the VLPs of the population retain their quaternary structure.
- the HEV VLP can be delivered to a subject via an oral route and the efficiency of delivery assessed by detecting and/or quantifying: (i) an immune response to an antigen within the HEV VLP; (ii) a detectable label conjugated to, recombinantly introduced into, or encapsulated by the HEV V LP; or (iii) a biological response due to delivery to a cell of a bioactive agent associated with (e.g., recombinantly introduced into, conjugated to, or encapsulated by) the HEV VLP.
- a bioactive agent associated with e.g., recombinantly introduced into, conjugated to, or encapsulated by
- an acid and proteolytical!y stable modified HEV VLP refers to a modified HEV VLP that retains at least 10%, 25%, 50%, 75%, 90%, 95%, 99%, or 100%) of the oral delivery efficacy and/or cell entry activity of an unmodified HEV V LP.
- heterologous refers to the two elements such as nucleic acids (e.g. , promoter or protein encoding sequence) or proteins (e.g. , an HEV ORF2 protein, or portion thereof, or modified capsid protein and another protein) that are not naturally found in the same relative positions.
- a heterologous promoter of a gene refers to a promoter that is not naturally operably linked to that gene.
- Hepatitis E virus is known to cause severe acute liver failure.
- HEV belongs to the genus Hepevirus in the family Hepeviridae.
- HEV contains a single-stranded positive- sense R.NA molecule of approximately 7.2-kb.
- the RNA is 3' polyadenylated and includes three open reading frames (ORE).
- ORF1 encodes viral nonstructural proteins, located in the 5' half of the genome.
- ORF2 encodes a protein-forming viral capsid, located at the 3' terminus of the genome.
- ORF3 encodes a 13.5-kDa protem, overlapped with C-terminus of ORF1 and N-terminus of ORF2.
- ORF3 is associated with the membrane as well as with the
- encapsulation refers to the envelopment of a heterologous substance, such as a heterologous nucleic acid or protein, a chemotherapeutic, an imaging agent, a ferrite nanoparticle etc., within the VLPs defined herein.
- bioactive agent refers to any agent, drug, compound, or mixture thereof that targets a specific biological location (targeting agent) and/or provides some local or systemic physiological or pharmacologic effect that can be demonstrated in vivo or in vitro.
- Non-limiting examples include drugs, hormones, vaccines, antibodies, antibody fragments, vitamins and co factors, polysaccharides, carbohydrates, steroids, lipids, fats, proteins, peptides, polypeptides, nucleotides, oligonucleotides, polynucleotides, and nucleic acids (e.g., mRNA, tRNA, snR A, R Ai, DNA, cDNA, antisense constructs, ribozymes, etc.).
- a "pharmaceutically acceptable” or “pharmacologically acceptable” material is one that is not biologicall harmful or otherwise undesirable, i.e., the material may be
- excipient refers to any essentially accessory substance that may be present in the finished dosage form of the composition of this invention.
- excipient includes vehicles, binders, disintegrates, fillers (diluents), lubricants, glidants (flow enhancers), compression aids, colors, sweeteners, preservatives,
- suspending/dispersing agents film formers/coatings, flavors and printing inks.
- adjuvant refers to a compound that, when administered in conjunction with an antigen, augments the immune response to the antigen, but does not generate an immune response to the antigen when administered alone.
- adjuvants can augment an immune response by several mechanism including lymphocyte recruitment, stimulation of B and /or T cells, and stimulation of macrophages.
- An "immunogenic response" to an antigen or composition is the development in a subject of a humoral and/or a cellular immune response to an antigen present in the composition of interest.
- a “humoral immune response” refers to an immune response mediated by antibody molecules
- a “cellular immune response” is one mediated by T-lymphocytes and/or other white blood ceils.
- CTL cytolytic T- cells
- CTLs have specificity for peptide antigens that are presented in association with proteins encoded by the major histocompatibility complex (MHC) and expressed on the surfaces of cells.
- helper T-cells help induce and promote the destruction of intracellular microbes, or the lysis of cells infected with such microbes.
- Another aspect of cellular immuni ty involves an antigen- specific response by helper T-cells.
- Helper T-cells act to help stimulate the function, and focus the activity of, nonspecific effector cells against cells displaying peptide antigens in association with MHC molecules on their surface.
- a "cellular immune response” also refers to the production of cytokines, chemokines and other such molecules produced by activated T-cells and/or other white blood cells, including those derived from CD4+ and CD8+ T-cells.
- an immunological response may include one or more of the following effects: the production of an tibodies by B-celis; and/or the acti vation of suppressor T-celis and/or ⁇ T-cells directed specifically to an antigen or antigens present in the composition or vaccine of interest.
- These responses ma serve to neutralize infeetivity, and/or mediate antibody-complement, or antibody dependent cell cytotoxicity (ADCC) to provide protection to an immunized host.
- ADCC antibody dependent cell cytotoxicity
- Such responses can be determined using standard immunoassays and neutralization assays, w r ell known in the art.
- the term "host” refers to humans as well as other animals.
- mucosal delivery relates to delivery of a composition to a mucous membrane, such as the mucosa of the gastrointestinal tract (e.g., the buccal or labial mucosa) or the mucosa of the respiratory tract (e.g. , the nasal mucosa).
- a mucous membrane such as the mucosa of the gastrointestinal tract (e.g., the buccal or labial mucosa) or the mucosa of the respiratory tract (e.g. , the nasal mucosa).
- One aspect of the invention relates to methods for production and purification of capsid proteins and VLPs derived therefrom (See, Expression and self-assembly of empty virus-like particles of hepatitis E virus.
- Li TC Yamakawa Y, S uzuki K, Tatsumi M, Razak MA, Uchida T, Takeda N, Miyamura T., J Virol. 1997 Oct;71(10):7207-13.
- Li TC Takeda N, Miyamura T, Matsuura Y, Wang JC, Engvail H, Hammar L, Xing L, Cheng RH. J Virol 2005 Oct;79(20): 12999-3006.
- Niikura M et al Chimeric recombinant hepatitis E virus-like particles as an oral vaccine vehicle presenting foreign epitopes.
- the capsid proteins are modified capsid proteins and the VLPs derived therefrom are cysteine modified HEV VLPs.
- the modified capsid proteins contain one or more cysteine residues in a surface variable loop of HEV OR 1 2. or a portion thereof.
- Various expression systems can be used to express the capsid proteins of the present invention.
- Examples of expression systems useful for the production of vims- like particles of the present invention include, but are not limited to, bacterial expression system (e.g.,
- Preferred expression system of the present invention includes baculovirus expression systems using insect cells.
- General methods for example, for handling and preparing baculovirus vectors and baculoviral DNA, as well as insect cell cul ture procedures, are outlined in A Manual of Methods for
- capsid proteins of the present invention can be cloned into the baculovirus vector, and used to infect appropriate host cells (see, for example, O'Reilly et al,
- An insect cell line ⁇ e.g., Sf9 or Tn5
- a transfer vector containing polynucleic acids which encodes the capsid proteins of the in venti on.
- Th e tran sfer vector in cludes, for example, linearized baculovirus DNA and a plasmid containing the desired polynucleotides.
- the host cell line may be co-transfected with the linearized baculovirus DNA and a plasmid in order to make a recombinant baculovirus.
- the modified capsid proteins or VLPs derived therefrom can be chemically conjugated to one or more bioactive agents.
- one or more cysteine residues of the capsid proteins can be acyiated, alkylated, aryiated, succinyiated, or oxidized using methods known in the art.
- the one or more cysteine residues can be conjugated using a maleimide functional group to covalentiy conjugate a bioactive agent to the thiol moiety of the cysteine, in some cases, the bioactive agent can be modified to introduce a maleimide functional group using CLICK chemistry.
- an alkyne deri vative of the bioactive agent can be contacted with a maieimide-azide in the presence of C11SO4 and ascorbic acid to produce a maleimide bioactive agent.
- the maleimide can then be contacted with the one or more cysteines of the modified capsid protein to covalentiy link the two molecules.
- the conjugating is performed on capsid protein that is not assembled into a VLP (e.g. , in the presence of EDTA, EGTA, and/or a reducing agent such as DTT or betamercaptoetha.no i).
- the conjugating is performed on capsid protein that is assembled into a VLP.
- HEV virus-like particles e.g., cysteine modified HEV VLPs
- Any standard technique in the art can be used to encapsulate a heterologous nucleic acid, protein, polypeptide, chemotherapeutic, imaging agent, nanoparticle, etc.
- the general proceed ure involves (1) disassembling the VLPs formed by the capsid protein according to the present invention; and (2) reconstructing the VLPs in the presence of the bioactive agent.
- a skilled artisan would recognize that it is preferred to have purified VLPs before the encapsulation procedure. It is particularly preferred to have the VLPs depleted of, or substantially depleted of, any undesired materials (e.g., nucleic acids) before the encapsulation procedure.
- Disassembly of VLPs can be carried out using any standard technique in the art. Reconstituted virus-like particle can be produced under physiological conditions (See, US Patent Publication No.: 20080131928). Often, disassembly of virus-like particles requires an agent to disrupt the assembly of VLPs, such as a reducing agent or a chelating agent (See, US Patent Publication No.: 20040152181). A skilled artisan would recognize that factors and conditions that affect assembly and disassembiy include: pH, ionic strength, posttranslational modifications of viral capsid proteins, disulfide bonds, and divalent cation bonding, among others.
- the VLPs of the present invention is disassembled by the removal of calcium ions (See, Touze A, Coursaget P. in vitro gene transfer using human papillomavirus- like particles. Nucleic Acids Res 1998; 26: 1317-1323; Takamura et a!., DNA vaccine- encapsulated virus-like particles derived from an orally transmissible virus stimulate mucosal and systemic immune responses by oral administration. Gene Therapy 2004; 1 1 :628-635).
- a reducing agent or a chelating agent or both are used to disassemble the VLPs.
- Various reducing agents can be used.
- Preferred embodiments of the reducing agents include, but are not limited to, dithiothreitol (DTT).
- DTT dithiothreitol
- Various chelating agents can be used, e.g., ethylene glycol tetraacetic acid (EGTA) or ethyl enediarninetetraacetic acid (EDTA).
- Examples of VLP disassembly conditions include, but are not limited to, the following: purified VLPs were disrupted by incubation of a buffer containing 50 mM Tris- HC1 (pH 7.5), 150 mM NaCf, 1 mM EGTA and 20 mM dithiothreitol for 30 minutes.
- VLPs complete disassembly of the VLPs is not required, although preferred, to encapsulate a bioaciive agent.
- An artisan would also recognize that, on other occasions, it is preferred to have partial disassembly of the VLPs.
- the conditions for the partial disassembly of the VLPs can be controlled to still allow efficient encapsulation of a bioaciive agent.
- Partial disassembly of the VLPs can be achieved by treatment of VLPs with reducing agents alone (e.g., 20 mM DTT) (Sapp et al, j . Gen. Virol., 76:2407-2412, 1995.).
- encapsulation of a bioactive agent can be carried out by reassembling the VLPs in the presence of the bioactive agent.
- a bioactive agent having a net negative charge it can be advantageous to utilize a bioactive agent having a net negative charge to enhance encapsulation.
- nucleic acids have a net negative charge and can be preferentially encapsulated as compared to compounds that have a positive or neutral charge.
- reassembly of the VLPs is achieved by re-supplementation of calcium ions to the disrupted V LPs.
- reassembly of the V LPs is achieved by removal of the reducing agents or the chelating agents.
- factors such as pH and ionic strength, other factors described in the present inven tion, can be adjusted to achieve effi cient reassembly of the VLPs and efficien t encapsul ation of the bioactive agent.
- encapsulation is performed as follows: Following 30 mm of incubation at room temperature, a bioactive agent in 50 mM Tris-HCl buffer (pH 7.5) and 150 mM aCl is added to the disrupted VLP preparation . The disrupted VLP preparation is then refolded by incubation for 1 h with increasing concentrations of Ca(3 ⁇ 4 up to a final concentration of 5 mM. VLPs are pel leted by ultracentrifugation and resuspended in 10 mM potassium-MES buffer (pH 6.2).
- VLPs are purified from any unencapsulated bioactive agent and disrupted with EGTA (1 mM). Absorbance of the supernatant, or other suitable methods can be used for detection of the bioactive agent,
- the bioactive agent or imaging agent to be encapsulated is conjugated to an encapsidation signal.
- an RNA element corresponding to codons 35-59 of HEV open reading frame 1 is a powerful encapsidation signal, allowing specific interaction in vitro with HEV capsid protein, including truncated and/or cysteine modified versions of HEV ORF2 VLP as described herein.
- VLP as a carri er for therapeutic or imaging agents
- chemical linkers e.g., LC-SPDP or aptamer, teiodendrimers
- tag the agent ⁇ e.g., chemotherapeutic with an HEV encapsidation signal like the foregoing RNA element can be used prior to the capsid self-assembly.
- a detectable label (imaging agent) is encapsulated.
- the detectable label can be a moiety renders a molecule to which it is attached to detectable by a variety of mechanisms including chemical, enzymatic, immunological, or radiological means.
- detectable labels include fluorescent molecules (such as fluorescein, rhodamine, Texas Red, and phycoerythrin) and enzyme molecules (such as horseradish peroxidase, alkaline phosphatase, and ⁇ galactosidase) that allow detection based on fluorescence emission or a product of a chemical reaction catalyzed by the enzyme.
- Radioactive labels involving various isotopes can also be attached to appropriate molecules to enable detection by any suitable methods that registers radioactivity, such as autoradiography. See, e.g., Tijssen, "Practice and Theory of Enzyme immunoassays," Laboratory Techniques in Biochemistry and Molecular Biology, Burdon and van Knippenberg Eds., Elsevier (1985), pp. 9 20.
- a bioactive agent is encapsulated.
- the bioactive agent is a chemotherapeutic.
- Suitable chemotherapeutics include, but are not limi ted to, cytotoxic drugs.
- cytotoxic drugs whi ch may be used in the present invention include: alkylating drugs, such as cyclophosphamide, ifospfamide, chlorambucil, meiphalan, busulfan, lomustine, carmustine, chlormethhine (mustine), estramustine, treosulfan, thiotepa, mitobronitol; cytotoxic antibiotics, such as doxorubicin, epirabicin, aciarubicin, idarubicin, daunorubicin, mitoxantrone (mitozantrone), bleomycin, dactinomycin and mitomycin; antimetabolites, such as methotrexate, capecitabine; cytarabine, fludarabine, cladribine, gemcitabine, fluorouracil, raltitrexed (tomudex
- one or more of the foregoing imaging agents and/or bioactive agents, or a combination thereof can additionally or alternatively be conjugated to a cysteine (e.g., recombinantly introduced cysteine) in a P-domain surface variable loop or C-terminus vi a a thiol, linkage.
- a cysteine e.g., recombinantly introduced cysteine
- one or more of the foregoing imaging agents and/or bioactive agents, or a combination thereof can additionally or alternatively be conjugated to a second cysteine ⁇ e.g. , recombinantly introduced cysteine) in a P-domain surface variable loop or C-terminus via a thiol linkage.
- the size of the VLPs can vary when different constructs of the capsid protein are used.
- the N-tenninal portion of the capsid protein can be adjusted to increase or decrease the size and encapsulation capacity of the VLPs.
- a portion of HEV ORF 3 protein fused to the N- terminal of a portion of HEV ORF 2 proteins is utilized to adjust the size of the V LPs.
- the HEV VLP is formed from a portion of HEV ORF2 having at least residues 1 12-608 of HEV ORF 2.
- the present invention also provides pharmaceutical compositions or physiological compositions comprising a cysteine modified HEV VLP formed by the capsid protein of the present invention.
- Such pharmaceutical or physiological compositions also include one or more pharmaceutically or physiologically acceptable excipients or carriers.
- Pharmaceutical compositions of the invention are suitable for use in a variety of drug delivery systems.
- Suitable formulations for use in the present invention are found in Remington 's
- compositions of the present invention can be administered to a host with an excipient.
- Excipients useful for the present invention include, but are not limited to, vehicles, binders, disintegrants, fillers (diluents), lubricants, giidants (flow enhancers), compression aids, colors, sweeteners, preservatives, suspending dispersing agents, film formers/coatings, flavors and printing inks.
- adjuvants can be used to increase the immunological response, depending on the host species, and include but are not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacender Calmette Guerin) and Corynebacterium parvum. Such adjuvants are also well known in the art. Further adjuvants that can be administered with the compositions of the invention include, but are not limited to, Monophosphoryl. lipid immunomodulator, AdjuVax 100a, QS 21 , QS 18,
- compositions and methods of the present invention can be administered to stimulate immune response without an adjuvant. Therefore, the compositions of the present invention are in some cases administered to a host without an adjuvant.
- compositions of the present invention are suitable for oral delivery. Because mucosal surfaces are inter-connected, stimulation of one mucosal surface by an antigen can induce mucosal immunity not only on the directly stimulated surface, but also on the distant ones.
- oral delivery of the compositions of the present invention can protect against respiratory and genital -urinary infections.
- the compositions of the present invention are also suitable for mucosal delivery, such as delivery to the buccal or labial mucosa or the respiratory tract mucosa, including the nasal mucosa.
- the pharmaceutical composi tions of the present invention can be administered by various routes, e.g.
- oral subcutaneous, transdermal, intradermal, intramuscular, intravenous, or intraperitoneal .
- the preferred routes of administering the pharmaceutical compositions are oral deliver ⁇ ' at daily doses of about 0.01-5000 nig, preferably 5-500 mg.
- the appropriate dose may be administered in a single daily dose or as divided doses presented at appropriate intervals, for example as two, three, four, or more subdoses per day.
- inert and pharmaceutical ly acceptable carriers are used.
- the pharmaceutical carrier can be either solid or liquid.
- Solid form preparations include, for example, powders, tablets, dispersible granules, capsules, cachets, and suppositories.
- a solid carrier can be one or more substances that can also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, or tablet disintegrating agents; it can also be an encapsulating material .
- the carrier is generally a finely divided solid that is in a mixture with the finely divided active component, e.g., a chimeric virus-like particles with an encapsulated nucleic acid.
- the active ingredient a chimeric virus-like particles with an encapsulated nucleic acid
- the carrier having the necessary binding properties in suitable proportions and compacted in the shape and size desired.
- a low- melting wax such as a mixture of fatty acid glycerides and cocoa butter is first melted and the active ingredient is dispersed therein by, for example, stirring. The molten homogeneous mixture is then poured into convenient-sized molds and allowed to cool and solidify.
- Powders and tablets preferably contain between about 5% to about 70% by weight of the active ingredient.
- Suitable carriers include, for example, magnesium carbonate, magnesium stearate, talc, lactose, sugar, pectin, dextrin, starch, tragacanth, methyl cellulose, sodium carboxymethyl cellulose, a low-melting wax, cocoa butter, and the like.
- compositions can include the formulation of the active compound with encapsulating material as a carrier providing a capsule in which the active component (with or without other carriers) is surrounded by the carrier, such that the carrier is thus in association with the compound, in a similar manner, cachets can also be included. Tablets, powders, cachets, and capsules can be used as solid dosage forms suitable for oral administration.
- Liquid pharmaceutical compositions include, for example, solutions suitable for oral or parenteral administration, suspensions, and emulsions suitable for oral administration.
- Sterile water solutions of the active component e.g., a chimeric virus-like particles with ars encapsulated nuc leic acid
- solvents comprising water, buffered water, saline, PBS, ethanol, or propylene glycol
- the compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, detergents, and the like.
- Sterile solutions can be prepared by suspending the active component (e.g., a chimeric virus-like particles with an encapsulated nucleic acid) in the desired solvent system, and then passing the resulting solution through a membrane filter to sterilize it or, alternatively, by dissolving the sterile compound in a previously sterilized solvent under sterile conditions.
- the resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous carrier prior to administration.
- the pH of the preparations typically will be between 3 and 9, more preferably from 5 to 8, and most preferably from 6 to 7.
- compositions of the present invention can be administered for prophylactic and/or therapeutic treatments.
- compositions are administered to a patient already suffering from a condition in an amount sufficient to prevent, cure, reverse, or at least partially slow or arrest the symptoms of the condition and its complications.
- An amount adequate to accomplish this is defined as a "therapeutically effective dose.” Amounts effective for this use will depend on the severity of the disease or condition and the weight and general state of the patient, but generally range from about 0.1 mg to about 2,000 mg of the composition per day for a 70 kg patient, with dosages of from about 5 mg to about 500 mg of the composition per day for a 70 kg patient being more commonly used.
- compositions of the present invention are administered to a patient susceptible to or otherwise at risk of developing a disease or condition, in an amount sufficient to delay or prevent the onset of the symptoms.
- an amount is defined to be a "prophylactically effective dose.”
- the precise amounts of the composition again depend on the patient's state of health and weight, but generally range from about 0.1 mg to about 2,000 mg of the inhibitor for a 70 kg patient per day, more commonly from about 5 mg to about 500 mg for a 70 kg patient per day,
- compositions can be carried out with dose levels and pattern being selected by the treating physician.
- pharmaceutical formulations should provide a quantity of composition of the present invention sufficient to effectively stimulate immune response in the patient, either therapeutically or prophylatically.
- VLPs Virus-like particles
- This application relies mainly on the property of self-assembly as well as the ease of genetic modifications, to fulfill the designed application for the given VLP.
- chemical conjugation of foreign peptides to VLP displays a significant advantage because it al lows a great variety of entities, such as peptide or oligosaccharide, to be conjugated to the surface of vims like particles in modulated and flexible manner without alteration of VLP assembly.
- Hepatitis E virus is composed of a non-enveloped icosahedral capsid, enclosing a single stranded RNA genome of 7.2 kilo bases.
- the major capsid protein is encoded by the second open reading frame (ORF2) and is essential not only for virus assembly, but also for immunogenicity and host interaction. 6
- the recombinant capsid protein (PORF2) is able to self-assemble into VLPs when expressed in insect cells after deletion of 111 amino acids from the N -terminal end and 52 amino acids from the C-terminal end.
- PORF2 folds into three domains: S (shell; amino acids 1 18-317), M (middle; amino acids 318-451) and P (protruding; amino acids 452-606).
- ⁇ 11 Recombinant HEV-VLPs consists of 60 copies of PORF2, in which the S-domain possesses typical eight anti-parallel folding and stabilizes icosahedral shell and the P-domain protrudes as a surface spike that contributes greatly to HEV antigenicity.
- HEV-VLP can be readily manipulated to, e.g., alter antigenicity, reduce the size from a T3 icosahedral particle to a Tl icosahedral particle, and/or modify the sequence at the P-domain without interfering with HEV-VLP assembly.
- HEV- VLP can be stable in acidic environment' ' and resistant to proteolytic digestion, 13 thus it poses a great advantage as an oral delivery vehicle.
- oral administration of HEV- VLP elicits both systemic and mucosal immunity without eliciting tolerance, and thus can provide protective immunity against HEV challenge in non- human primates 15
- Chimeric VLPs carrying a foreign epitope are able to elicit mucosal and systemic antibodies against both HEV and the foreign epitope after oral administration.
- HEV-VLP can orally deliver plasmid DNA to the epithelial cells of the small intestine and induce antibody and cytotoxic T lymphocyte (CTL) responses against the plasmid encoded antigen.
- CTL cytotoxic T lymphocyte
- the modified HEV-VLP after conjugation with LXY30, a ligand peptide with a high affinity for human malignant breast tumor cells, 58 showed specific targeting to breast tumor ceil both in vitro and in vivo, indicating that delivery route of viruslike particle can be manipulated to facilitate targeted delivery of chemotberapeutic drugs or siRNA to pathologic foci.
- Aikyiation and acylation are the two traditional bioconjugation methods that are used to covendedly modify virus particles by attaching a ligand at amine group of lysine or thiol group on cysteine, respectively.
- HEV ORF2 naturally has four lysine residues that are
- the P-domain carries the antigenic structure of HEV
- sequence mutation may alter the immunoreactivity of HEV antibodies against the obtained chimeric VLPs.
- the antigenicity of the chimeric N573C VLP to the wild type HEV- VLP in the presence or absence of biotin conjugation was compared.
- An ELISA plate was coated with N573C VLP without conjugation, maleimide-biotin conjugated N573C VLP, or the wild type HEV-VLP and then incubated with two monoclonal antibodies, HEP230 or HEP40-4.
- HEP230 In contrast to HEP40-4, HEP230 exhibited strong binding for the wild type HEV-VLP, but failed to exhibit detectable binding to N573C VLP regardless of biotin conjugation (Fig 2B). The ELISA against the mixture of HEP230 and HEP40-4 showed similar trend (data not shown). This data suggested that residue N573 is important to HEP230 binding. Residue N573 is Involved in FA.B230 Binding
- connection density between the spikes was much weaker than the capsid and was not big enough to accommodate the Fab molecule.
- the size of the connection density appeared to be in good agreement with the contacting surface of the Fab molecule (Fig 3B), indicating that the Fab230 may contact VLP at this position.
- LXY3Q Conjugated to N573C VLP Shows Cancer Cell Targeting [ ⁇ 090]
- a breast cancer cell targeting ligand L.XY30
- L.XY30-VLPs The LXY30-decorated N573C-VLPs (LXY30-VLPs) remained intact after the chemical conjugation (Fig. 4).
- Flow cytometry revealed a significantly higher attachment (>5 fold) for LXY30-VLPs to MDA-MB-231 breast cancer cells, compared to that for the wild type VLPs (Fig.
- LXY30-VLPs enter the MDA-MB-231 cells.
- the LXY30-VLPs were labeled with the near infrared dye, Cy5.5, and incubated with MDA-MB-231 cells at 37 °C for 1 hour.
- Fig 5B most Cy5.5-labeled VLP distributed around the perinuclear region of MDA-MB-231 cells, which meant the VLP was internalized into the cytoplasm.
- LXY30 and scrambled LXY30 was also tested for uptake by the glioma cell line U-87MG, suggesting its use as a conjugate for HEV -VLP for targeting to glioma ceils (Fig. 6).
- Noninvasive in vivo imaging has been shown to be a powerful tool to visually monitor the delivery of therapeutic reagents.
- Both Cy5.5 labeled LXY30-VLPs and the Cy5.5 labeled wild type VLPs were intravenously injected via tail vein in nude mice bearing DA-MB-23.1 xenografts, respectively. The mice were scanned with the fluorescence imager at various intervals for up to 72 hours.
- HEV as an enteric transmitted virus, preferentially enters hepatocytes in liver, it is not surprising that Cy5.5 labeled VLPs accumulated within one hour at the abdominal organs including liver. Ex vi vo imaging of excised organs were performed at 72 hours post injection. The remaining signal of Cy5.5 was found predominantly at the liver and kidney, however, the signal appeared weak in excised tumor (data not shown). The high uptake in liver and kidney could be explained in part as leaking of the breakdown VLPs from circulating vessels. Overall, these results indicate that Cy5.5/LXY30 linked HEV- VLPs is able to target MDA-MB-231 xenografts in vivo.
- VLPs As a drug delivery system for diseases that require the delivery of cytotoxic drugs or specific genetic modifications, ceil tagging, or drugs that require encapsulation .
- VLPs provide an opportunity for tissue/cell specific approaches while minimizing the overall damage to healthy cells.
- HEV-VLP like the Hepatitis E vims can maintain its structure through the low pH and proteolytic
- HE V-VLPs could have a higher propensity for efficient delivery and reduced
- the streptavidin homotetramer can intercalate into the surface depression and form multivalent interaction with N573C anchored biotin molecules, which largely reduces the rate of dissociation.
- the avidity of streptavidin to N573C VLP is the highest although the affinity of streptavidin to a single biotin appeared to be the same as it showed by its binding to the disassembled VLPs.
- the quaternary arrangement of cysteine residues in the other chimeric VLPs does not support multivalent binding of streptavidin.
- N573 residue is located within the binding footprint of Fab (Fig 3), so that mutation of N573 blocks the interaction of PORF2 and HEP2.30.
- the volume of a fivefold depression is not sufficient to s multaneously accommodate five Fab molecules, so that the structure of Fab-bound VLP can only capture the conta cting region of the Fab mo lecule after icosahedra!-average.
- the density is about 20% occupancy to that of the capsid, suggesting that only one Fab molecule resided at each five-fold axis. This is consistent with the non-icosahedral symmetry averaged density map.
- HEV-573C VLPs were chosen as a platform of modularized theranostic capsule because of its possibility of weakening the immunoresistance against the pre-existing HEV antibodies as well as the possibility of multi valent binding with the ligand.
- thiol-selective conjugation for coupling a cancer cell targeting ligand to VLP, because of the spatial specificity provided by cysteine replacement.
- the conjugation of dye was performed by amine-selective conjugation that gave rise to sufficient MR signal for us to detect VLP distribution in MDA-MB-231 breast cancer ceils in vitro and in vivo (in mice). Therefore, the HEV-Cys VLPs can be used as a platform for dual-functional, tagging with cancer adliesion-ligand and a detection marker, a robust candidate for cancer diagnosis.
- HEV-Cys- VLP is capable of encapsulation of a variety of bioactive substances.
- HEV-Cys- VLPs can encapsulate magnetic nano-particles such as ferrite, for both diagnosis under MR! and thermotherapy. in such cases, the magnetic nano- particies can be selectively stimulated using ultrasound or radio frequenc electromagnetic radiation to heat the ferrite particles encapsulated in the tumor targeted theroanostic capsule.
- Negatively charged micro-RNA/siRNA could be also encapsulated into the interior of HEV-Cys-VLPs. ' By binding the cancer cell targeting ligand on its surface, HEV-Cys-VLP could have potential applications in cancer cell targeting gene therapy;
- baculovirus-based expression vectors The recombinant baculoviruses used to express the mutated HEV ORF2 proteins were generated using the Bac-to-Bac® Baculovirus Expression System (In vitro gen) on Sf9 cells (Invitrogen) and each of the pFastBacl/ HE V ORF2 plasmids described above according to the protocols supplied by the manufacturer.
- the Sf9 and High Five cells were maintained on ESF921 medium (Expression Systems, Davis, CA USA) and Exceli 420 medium (SAFC Biosciences, Lenexa, KS) supplemented with 2.5% heat-inactivated FBS, respectively, following standard protocols. 24
- ESF921 medium Expression Systems, Davis, CA USA
- Exceli 420 medium SAFC Biosciences, Lenexa, KS
- FBS heat-inactivated FBS
- VLPs were collected and purified through multiple steps of CsCl equilibrium densit gradient ltracentrifugation as described previously.' The purified VLPs were resuspersded in 10 mM potassium-MES buffer, pH6.2, and stored at 4 °C. A Mini dialysis device (Miliipore) was used in the disassembly experiments. The purified VLPs were disrupted by dialysis against buffer containing EDTA (lOmM) or/and DTT (20mM).
- the purified VLPs were loaded onto a glow-discharged, carbon -coated EM grid and stained with 2% uranyl acetate and examined under a JOEL. JEM-1230 transmission electron microscope at a magnification of 3Q.00QX. The images were recorded on a CCD camera (TVIPS Gauting, Germany).
- PVDF polyvinyl! dene fluoride
- HRP conjugated streptavidin at a ratio of 1 : 10,000.
- the biotin- streptavidin reaction was then detected by an enhanced chemiluminescence method using an ECL kit (Amersham Biosciences, Piscataway, NJ).
- Both recombinant HEV -WT-VLP particles including HEV-WT and HEV-Cys mutants were prepared in 10 mM potassium-MES, pH 6.2 coating buffer.
- the proteins were diluted to the final concentrations of 1-100 ng/ml and coated overnight at 4 °C onto a clear bottom 96-well plate (Nunc, Pleasant Prairie, WI). Control wells were inclubated with 0.01 M PBS, pH 7.2.
- the coated VLPs were incubated with ⁇ 50 ⁇ of the HEV antibodies for two hours at 37 °C after blocking with 0.5% Tween-20 in Tris buffer containing 20 mM Tris, pH 7.4, and 150 mM NaCl.
- the associated HEV antibodies were detected using alkaline phosphatase-labeled anti-mouse FAb.
- the enzymatic reaction was developed using pnitrophenyiphosphate (pNPP) solution (Sigma Co).
- pNPP pnitrophenyiphosphate
- the yellow color product of nitrophenyi was measured at 405 ra using a microplate reader and the average absorbance value of each VLP was calculated.
- Maleirnide-conjugated LXY30 was prepared by mixing 650 ⁇ maleimide-azide and 650 ⁇ aikyne-LXY ⁇ 3Q in the presence of 200 ⁇ CuS04 and 1 mM ascorbic acid at 4 °C overnight. Then maleirnide-conjugated LXY30 was added at molar ratio of 3: 1 (ligand vs. binding site) to react with the C s residues on HEV-Cys mutants though a thiol reaction at 4 °C overnight. The unconjugated molecules were removed by a 7,000 MWCO desalting columns, (ZebaTM Spin Desalting Columns, Thermo Scientific).
- Cy5.5 NHS ester (200nm) was used to react with HEV-Cys mutants (20 ⁇ ) at molar ratio of 300: 1 (dye vs VLP) in buffer containing 0.01 M PBS, pH 7.2 at room temp for 2 hrs, following by incubation at 4 °C overnight.
- the free Cy5.5 NHS ester was then removed by a 7,000 MWCO desalting column, (ZebaTM Spin Desalting Columns, Thermo Scientific). Flow Cytometry
- MDA-MB-231 breast cancer cells were incubated with Cy5.5-labeled HEV-573C VLPs (VLP-Cy5.5) or Cy5.5/LXY30 conjugated HEV-573C VLPs (LXY3-VLP Cy5.5) for 1 hour at 37 °C, respectively. Then the cells were washed with PBS 3 times and re-suspended in PBS for the flow cytometric analysis. 1 A total of 0, 000 events were collected for each sample. Unstained ceils were used as a control.
- mice Female SPF BALB/c mice, 10-12 weeks age, were purchased from Charles River (Davis, CA)) and kept under pathogen-free conditions according to AAALAC guidelines and were allowed to acclimatize for at least 4 days prior to any experiments.
- DA-MB-231 breast cancer cells were injected subcutaneously into nude mice to form subcutaneous nodules. All animal experiments were performed in compliance with institutional guidelines and according to protocol No. 06-12262 approved by the Animal Use and Care
- mice were intravenously injected through the tail vein with 4 nmol/L Cy5.5 fluorescent-labeled HEV-573C VLPs (VLP-Cy5.5) and LXY30 conjugated HEV-573C VLPs (LXY30- VLP-Cy5.5) respectively.
- mice were scanned with Kodak imaging system IS20QQMM according to the procedure described previously." Images were collected with an excitation bandpass filter at 625 nm and an emission at 700 nm, exposure time was 30 s per image. After in vivo imaging, animals were euthanized by C0 2 overdose at 72 h after injection. Tumors, organs, and muscle tissue were excised and imaged with the Kodak imaging station. Preparation of VLP-Fab Complexes for Cryoelectron Microscopy
- VLP-Fab complexes were prepared by incubating VLPs and Fabs at a molar ratio of 1 : 180 at 4 °C overnight. To ensure optimal purity, maxima] occupancy, and ultimately reduced background noise during structural determination, pure VLP-FAB complexes were obtained by passing the sample through a short Sephacryl-300 gel -filtration column. Optical density readings at a wavelength of 280 nm were used to select the fractions that contained VLP-Fab complexes. The purified VLP fraction was then characterized by SDS-PAGE under reducing condition to validate the binding of Fab.
- Figure 10 depicts various routes for modifying the surface of HEV ORF2 VLP nanocarriers and encapsulation of detectable labels, drugs, or nucleic acid payloads.
- An HEV ORF2 VLP-based nanocarrier can self-assemble in vitro and encapsulate anticancer therapeutics or image reagents. With genetic engineering, cancer targeting iigands can be conveniently ligated to the surface of the carrier for cancer-specific targeted delivery.
- the chimeric VLP exhibits excellent biocompacibility, high absorption of near- infrared light, and structure-dependent fluorescence quenching/emission.
- This nanomedicme platform allows integration of multiple imaging and therapeutic modalities for superior cancer detection and therapy.
- Intratumoral delivery is a challenge task for modem medical research and is a process that comprises lots of negotiations betw een nanocarriers with cells, extracellular matrix, and the vasculature inside the tumor.
- Described herein is an HEV -VLP based nanocarrier for targeted tumor delivery guided by imaging the in situ distribution of VLP at the whole animal level, to the tumor interior, and ultimately down to subcellular organelles.
- Engineered HEV-VLPs for tumor targeting are provided by adding adhesion tag(s) to the surface of the P-domain.
- a genetical ly inserted a fluorescence marker can also be inserted into the VLP so that it can be used as endogenous reporter.
- Use of these nanoparticles demonstrate the power of correlative microscopic studies on the role it plays in the development of nanocarriers.
- Nanocarriers composed of synthetic polymers, liposomes, and dendrimers, have been well-investigated, however, there are only two FDA-approved antibody-conjugates and four FDA-approved nanoparticle-based drug delivery carriers.
- Virus like particle is natural provided nanocarriers that possess several attractive features. They can be self-assembled into monodisperse, nansized particles, with defined periodicity that enables dense, repetitive display of tumor targeting agents. In addition, the V LP possesses multiple unique features that make it more attractive as potential nanocarrier.
- HEV VLP nanoparticles can be characterized with electron microscopy in the presence or absence of contrast enhancing reagents. Microscopic characterization of nanoparticles can be routinely performed to examine the physical properties of the nanocarriers using methods described herein, such as imaging frozen hydrated
- multifunctional particles e.g. , using contrast enhanced gold atom clusters (Fig. 1 1A).
- Methods and compositions for conjugating gold clusters to cysteine thioiates include direct ligand exchange and/or maleimide cysteine conjugation (see, e.g., Ackerson ei al, Bioconjug. Chem., 21 , 214-218 (2010); Ackerson et al., Methods EnzymoL, 481 : 195-230 (2010); and Maijormaki et al, PNAS, 11 1(4): 1277 ⁇ 81 (2014)).
- the direct visualization by electron microscopy precisely reveals the size, shape, and domain arrangement of the nanoparticles, as well as the protein/drug absorption on nanoparticles, factors that can influence the overall Inactivity of the nanoparticles and the efficacy of targeted tumor delivery .
- the direct visualization by electron microscopy precisely reveals the size, shape, and domain arrangement of the nanoparticles, as well as the protein/drug absorption on nanoparticles, factors that can influence the overall Inactivity of the nanoparticles and the efficacy
- nanoparticles can be imaged with either by cryo-electron microscopy on unstained frozen hydrated specimen or by traditional electron microscopy on negatively stained specimen.
- the cryoEM is well-suited to imagine the biological macromolecular complexes (Fig. 11 A- D).
- correlative microscopic techniques ca be based on these newl described conjugates to achieve the utilization of multiple imaging modalities in the examination of the same tumor specimen in order to deliver information above and beyond the capability of either modality alone.
- li ght and electron microscopy can be used to image from the organismal level, to the organ level, to cells, and even subcellular details.
- MR Near-Infrared
- CLEM correlative light and electron microscopy
- VLP intratumorai distribution With, e.g., high pressure freezing and freeze substitution, tumor diagnosis can be preserved to maintain the sufficient antigenicity of HEV-VLP for immunofluorescence labeling.
- Region identification is one of the critical issues for tumor diagnosis, where both light and electron microscopies indeed image the same object.
- the coordinates of 1 ) photoconversion of DAB (3,3 '-diammobenzidine tetrahydrochrloride); 2) dual functional probe, FluoroNanogold; and 3) EM finder grids can be mformative in multiple modalities of tumor identification.
- the first method uses free oxygen radicals that formed upon illumination of fluorochromes to induce oxidation of DAB in situ.
- FluoroNanogold conjugated antibody is a commercially available reagent that provides both light and electron microscope contrast by conjugation of 1 ,4 nm gold clusters either with green or with red fluorophore. Unlike colloidal gold of other sizes, the 1.4 nm nanogold cluster does not quench fluorescence.
- HEV VLPs described herein allow detection and visualization by 3D electron tomography to monitoring drug delivery.
- Electron tomography provides 3D spatial information to describe the biodistribution of drug delivery vehicles inside the tumor. In a three-dimensional entity, the microenvironment of tumor can be much more complex than what found in vitro with cultured monolayer cells. Electron tomography can be used as a complement to C LEM in analyzing the intratumoral accumulation of drug- loaded V LP, so as to elucidate the mechanism for VLP extravasation and passing through the cell layers inside a tumor.
- HEV VLPs can be equipped and encapsulated with additional imaging agents, e.g., water-soluble CdSe/ZnS quantum dots or Ferrite, in a disassembly-and-reassembly process (Fig 12).
- additional imaging agents e.g., water-soluble CdSe/ZnS quantum dots or Ferrite
- Quantum dots can provide an dual-functional probe to track VLP tumor uptake with correlative light and electron microscopy.
- the efficiency of packaging can be optimized to have efficient encapsulation, e.g., by coating the quantum dots with an HEV encapsidation signal through LC-SPDP- mediated crosslinking.
- RNA element occupying codons 35-59 of HEV open reading frame 1 is a powerful encapsidation signal, allowing specific interaction in vitro with HEV capsid protein, including truncated and/or cysteine modi fied versions of HEV ORF2 VLP as described herein.
- VLP as drag carrier, chemical linkers (e.g., LC-SPDP or aptamer, telodendrimers) that tag the drug (e.g., chemotherapeutic) with an HEV encapsidation signal like the foregoing RNA element can be used prior to the capsid self-assembly.
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